Hydrogen producing activity by Escherichia coli hydrogenase 4 (hyf) depends on glucose concentration

Abstract

Escherichia coli produces molecular hydrogen (H2) during glucose fermentation. This production of H2 occurs via multiple and reversible membrane-associated hydrogenases (Hyd). Dependence of H2 producing rate (VH2)(VH2) by Hyd-4 (hyf ) on glucose concentration was studied at different pHs. During growth on 0.2% glucose at pH 7.5 in JRG3615 (hyfA-B ) and JRG3621 (hyfB-R ) mutants (VH2)(VH2) was decreased ∼6.7 and ∼5 fold, respectively, compared to wild type. Only in JRG3621 mutant at pH 6.5 and 5.5 (VH2)(VH2) was severely decreased ∼7.8 and ∼3.8 fold, respectively. But when cells were grown on 0.8% glucose no difference between wild type and mutants was detected at any of the tested pHs. The results indicate Hyd-4 H2 producing activity inhibition by high concentration of glucose mainly at pH 7.5. This is of significance to regulate Hyd activity and H2 production by E. coli during fermentation.

Full text

Short Communication
Hydrogen producing activity by Escherichia coli
hydrogenase 4 (hyf) depends on glucose
concentration
K. Trchounian
a,b
, A. Trchounian
a,*
a
Department of Microbiology&Plants and Microbes Biotechnology, Yerevan State University, 0025 Yerevan,
Armenia
b
Department of Biophysics, Yerevan State University, 0025 Yerevan, Armenia
article info
Article history:
Received 5 June 2014
Received in revised form
3 August 2014
Accepted 14 August 2014
Available online 4 September 2014
Keywords:
Low and high concentration of
glucose
Hydrogenase 4 (hyf)
pH
Hydrogen production
Escherichia coli
abstract
Escherichia coli produces molecular hydrogen (H
2
) during glucose fermentation. This pro-
duction of H
2
occurs via multiple and reversible membrane-associated hydrogenases (Hyd).
Dependence of H
2
producing rate ðVH2Þby Hyd-4 (hyf) on glucose concentration was studied
at different pHs. During growth on 0.2% glucose at pH 7.5 in JRG3615 (hyfA-B) and JRG3621
(hyfB-R) mutants ðVH2Þwas decreased ~6.7 and ~5 fold, respectively, compared to wild type.
Only in JRG3621 mutant at pH 6.5 and 5.5 ðVH2Þwas severely decreased ~7.8 and ~3.8 fold,
respectively. But when cells were grown on 0.8% glucose no difference between wild type
and mutants was detected at any of the tested pHs. The results indicate Hyd-4 H
2
pro-
ducing activity inhibition by high concentration of glucose mainly at pH 7.5. This is of
significance to regulate Hyd activity and H
2
production by E. coli during fermentation.
Copyright ©2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.
Introduction
Currently substantial mitigation of fossil fuels needs to find
cheap, ecologically clean alternative energy sources. One of
these sources is molecular hydrogen (H
2
) which can be pro-
duced from microbial and algal biomass [1].H
2
production
from different sugars or organic carbon-containing industrial,
agricultural, water and other kind wastes has been set on well
and the biotechnology has been already elaborated. Moreover,
co-fermentation of different carbon sources by different bac-
teria resulting H
2
production has been shown [2e5].
It is well known that H
2
is produced via special membrane-
associated enzymes named hydrogenases (Hyd) which
reversibly oxidize H
2
to 2H
þ
.Escherichia coli has the capacity to
encode four [NieFe]-hydrogenases, three of which are bio-
chemically and genetically characterized well [6] but the ac-
tivity of Hyd-4 (hyf) is described vague [7]. Hyd-3 (hyc) and Hyd-
4 with formate dehydrogenase H (FDH-H) are suggested to
form formate hydrogen lyase (FHL)-1 and FHL-2 pathways,
*Corresponding author. Tel.: þ374 60710520.
E-mail address: Trchounian@ysu.am (A. Trchounian).
Available online at www.sciencedirect.com
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international journal of hydrogen energy 39 (2014) 16914e16918
http://dx.doi.org/10.1016/j.ijhydene.2014.08.059
0360-3199/Copyright ©2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

respectively [6]. Hyd-1 (hya) and Hyd-2 (hyb) are reversible Hyd
enzymes which can operate in different mode depending on
carbon source: during glucose or glycerol fermentation they
operate in H
2
uptake or producing mode, respectively [6,8].
Thus, the important features of Hyd enzymes are their mul-
tiplicity and reversibility.
It was demonstrated that Hyd-4 is primarily active at
slightly alkaline pH and this enzyme is mainly responsible for
H
2
production by E. coli [9]. However, the conditions when
Hyd-4 activity is observed should be studied. Moreover, it was
detected that glucose has inhibitory effect on hyf genes
expression [10]. In addition, the stimulatory role of glucose on
phosphotransferase transport system in E. coli has been
established [11]. But the mechanism of glucose inhibitory ef-
fects on Hyd-4 is not clear.
Nowadays the advanced interest of H
2
production by E. coli
for developing H
2
bio-production technology is to detect and
to control the conditions of different enzymes activities. The
main goal is to demonstrate the effect of glucose concentra-
tion on H
2
producing activity of Hyd-4.
Materials and methods
Bacterial strains and growth
The E. coli MC4100 wild type and different hyf mutant strains
were used in this study (Table 1). Bacteria from an overnight
growth culture were transferred into the fresh liquid medium
(20 g L
1
peptone, 15 g L
1
K
2
HPO
4
, 1.08 g L
1
KH
2
PO
4,
5gL
1
NaCl) with different concentrations of glucose (0.2% or 0.8%) at
pH 5.5, 6.5 or 7.5. Overnight growth culture was the same as
fresh liquid medium at appropriate pH mentioned and at
37 C; 1 ml of overnight culture per 100 ml of fresh medium
was transferred. Bacteria were grown under anaerobic con-
ditions at 37 C for 18e22 h as described [4e6]. For anaerobic
conditions glass vessels with plastic press caps were used; O
2
and N
2
dissolved in liquid medium were bubbled out of the
media by autoclaving, after which the vessels were closed by
press caps. pH was determined by a pH-meter with a selective
pH-electrode (ESL-63-07, Gomel State Enterprise of Electro-
metric Equipment (GSEEE), Gomel, Belarus; or HJ1131B, Hanna
Instruments, Portugal) and adjusted using 0.1 M NaOH or HCl.
Analytical methods
H
2
production assays were done by redox potential (E
h
)
determination. The latter was done using a pair of redox,
titanium-silicate (TieSi) (EO-02, GSEEE) and platinum (Pt) (EPB-
1, GSEEE; or PT42BNC, Hanna Instruments, Portugal) elec-
trodes as described in details previously [4e6,9].H
2
production
rate ðVH2Þwas calculated as the difference between the initial
decreases in Pt- and TieSi-electrodes readings per time. It was
expressed as mV of E
h
per min per mg dry weight of bacteria.
This approach is close to the method employed by Fernandez
[12] and different groups [13e15] with a Clark-type electrode: a
correlation between E
h
and H
2
production was shown. Using
the Durham tube method [9],H
2
production during the growth
of E. coli was also estimated by the appearance of gas bubbles
in the test tubes over the bacterial suspension.
H
2
production by the cells grown on various concentrations
of glucose was assayed with either 0.2% or 0.8% glucose.
Preparation of whole cells for H
2
production assays was
described before [4e6,9]. The cells were washed with distilled
water and then transferred into the assays mixture (100 mM
Tris-phosphate buffer (appropriate pH) containing 0.4 mM
MgSO
4
, 1 mM NaCl and 1 mM KCl); glucose was supplemented.
The assays were performed in a thermo-stated chamber at
37 C; bacterial suspension in the closed vessel was mixed
with a magnetic stirrer bar. Dry weight of bacteria was
determined as described previously [4,9].
Agar, glucose, peptone, Tris (Carl Roths GmbH, Germany),
and the other reagents of analytical grade were used for bac-
terial growth and hydrogen production assays.
Each data point represented is averaged from independent
triplicate cultures; the standard deviations calculated as
described [4,9] are not more than 3% if they are not repre-
sented. The validity of differences between experimental and
control data is evaluated by Student's criteria (p)[16];p<0.01
or less if this is not represented, otherwise p>0.5 if the dif-
ference is not valid.
Results
H
2
production during 0.2% glucose fermentation by E. coli
wild type and Hyd-4 mutants at different pHs
During fermentative growth of E. coli in the presence of 0.2%
glucose and in the assays supplemented with 0.2% glucose at
pH 7.5 JRG3615 (hyfA-B) and JRG3621 (hyfB-R) mutant strains
(see Table 1) had H
2
production rate ðVH2Þ~6.7 fold and ~5 fold
less, respectively, than wild type cells (Fig. 1). These data are in
good conformity with previously obtained results [9]. In the
same conditions addition of 0.8% glucose in the assays had the
same effect on H
2
generation. At pH 6.5 in JRG3615 and
JRG3621 strains ðVH2Þwas decreased ~2.2 fold and ~7.8 fold,
respectively, compared to wild type (see Fig. 1). But at pH 5.5 in
JRG3621 strain it was decreased ~3.8 fold, compared to wild
type (see Fig. 1). These findings point out that at pH <7.0 only
the deletion of the most of Hyd-4 operon genes disturbs H
2
production, unlessðVH2Þof JRG3615 is ~3.5 fold higher than
Table 1 eCharacteristics of E. coli strains used.
Strains Genotype Source and reference
MC4100 araD139 D(argF-lac)U169 ptsF relA1 fib5301 rpsL150 S.C. Andrews (The University of Reading, Reading, UK) [9]
JRG3615
a
MC4100 D(hyfA-B)::spc S.C. Andrews [9]
JRG3621
a
MC4100 D(hyfB-R)::spc S.C. Andrews [9]
a
Resistant to spectinomycin.
international journal of hydrogen energy 39 (2014) 16914e16918 16915

that of JRG3621 strain (see Fig. 1). The decreased H
2
production
might be due to interaction between Hyd-4 and Hyd-3 and the
lacking major part of Hyd-4 which affects Hyd-3 activity. As it
has been shown [9], small subunit of Hyd-3, hycB, is required
for H
2
production at pH 7.5 when Hyd-4 is responsible for the
H
2
generation. The data with Hyd-4 mutants suggest that the
deletion of single gene has no clear effect on H
2
production
activity of Hyd-3 or Hyd-4 because of compensatory effects
between these Hyd enzymes [6], but deleting bunch of the
hyfB-R important genes, which might be involved in electron
transfer chain from FDH-H to Hyd-3 or Hyd-4 or protons from
the F
0
F
1
-ATPase to secondary transport systems, might
disturb the pathways and has strong impact on H
2
production
overall.
During fermentative growth of E. coli in the presence of
0.2% glucose but in the assays supplemented with 0.8%
glucose the same effects of Hyd-4 impact on H
2
generation
was observed (see Fig. 1). In this respect it is suggested that
supplementation of glucose concentration is important for H
2
formation and addition during the assays of different con-
centrations of the carbon source has no effect.
H
2
production during 0.8% glucose fermentation by E. coli
wild type and Hyd-4 mutants at different pHs
During growth of E. coli in the presence of 0.8% glucose but in
the assays supplemented with 0.2% glucose at pH 7.5 wild type
and mutants showed similar H
2
producing activity (Fig. 2). In
JRG3621 strain ðVH2Þwas lowered, compared to wild type, but
this decrease at pH 5.5 was not significant and had no any
effect (see Fig. 2): impact of Hyd-4 on H
2
production was
detected.
In the assays with 0.8% glucose in wild type and mutants
no any difference in ðVH2Þwas observed (see Fig. 2). Again, only
small decrease of V
H2
was detected at pH 5.5 (see Fig. 2).
Importantly, it was shown before [17] that N,N'-dicyclohex-
ylcarbodiimide (DCCD), known inhibitor of the F
0
F
1
-ATPase
[9], inhibits H
þ
efflux in JRG3621 mutant at pH 5.5. At the same
time, the rate of K
þ
uptake was markedly lower in hyfR and
hyfB-R but not in hycE or hyfA-B mutants; H
þ
transport was
lowered and sensitive to DCCD in hyf but not in hyc mutants.
The results pointed out the relationship of K
þ
uptake and the
F
0
F
1
-ATPase with Hyd-4 activity. Interestingly, novel option
for the expression of some hyf genes in E. coli grown at pH 5.5
has been proposed: it is possible that the hyfB-R genes
expressed under acidic conditions or their gene products
interact with the gene coding for the K
þ
uptake Kup protein or
directly with the Kup system [17].
The data obtained suggest that at 0.8% glucose concen-
trations no any Hyd-4 activity in H
2
production or other pro-
cesses can be detected (see Fig. 2). This explains why in
different groups no any Hyd-4 activity is detected [18]: they
use common recipes of media where glucose concentrations
are higher than 0.2% (e.g. glucose concentration in the
peptone medium, agar or others is 0.8e1%).
Moreover, excess of glucose in the cells can change the
fermentative metabolism, and it might be that Hyd-4 is
switching on when there are limited conditions for the cell.
In addition, an association of the F
0
F
1
-ATPase with second-
ary transport systems or key enzymes of fermentation has
been proposed under energy limited processes when trans-
fer of energy from ATPase to the other membrane protein
might lead to the work increasing efficiency of energy using
[19,20].
Discussion
In the current study the effect of glucose concentration on E.
coli H
2
producing activity of Hyd-4 was investigated. It was
shown that low concentration of glucose (0.2%) affects the
activity of Hyd-4 and its involvement in H
2
production (see
Figs. 1 and 2). Besides, Self et al. [10] have described that, in LB
0
1
2
3
4
5
6
7
8
9
pH 7.5 pH 6.5 pH 5.5 pH 7.5 pH 6.5 pH 5.5
0.2% glucose 0.8% glucose
H2prodction rate, mV Eh/min/mg dry weight
MC4100
JRG3615
JRG3621
Fig. 1 eH
2
production rate by E. coli MC4100 wild-type,
JRG3615 (hyfA-B) and JRG3621 (hyfB-R) mutant strains
grown on peptone medium supplemented with 0.2%
glucose at different pHs. In the assays 0.2% or 0.8% glucose
were added as mentioned. For mutants see Table 1; for the
others details of culture conditions and H
2
assays see
Materials and methods.
0
1
2
3
4
5
6
pH 7.5 pH 6.5 pH 5.5 pH 7.5 pH 6.5 pH 5.5
0.2% glucose 0.8% glucose
H2prodction rate, mV Eh/min/mg dry weight
MC4100
JRG3615
JRG3621
Fig. 2 eH
2
production rate by E. coli MC4100 wild-type,
JRG3615 (hyfA-B) and JRG3621 (hyfB-R) mutant strains
grown on peptone medium supplemented with 0.8%
glucose at different pHs. For others see legends to Fig. 1.
international journal of hydrogen energy 39 (2014) 16914e1691816916

medium in the presence of glucose, expression of hyf operon
genes is inhibited. Moreover, hyfR, which was suggested by
Andrews et al. [7] as transcriptional activator for hyf operon,
was shown to activate hyf but to have no any effect on hyc
operon. Bagramyan et al. [9] have demonstrated that at
slightly alkaline pH Hyd-4 is major for H
2
production which
was confirmed by our data (see Fig. 1) but they also used low
concentration of glucose (0.2%). Interestingly, Mnatsakanyan
et al. [21] have detected that addition of external formate ac-
tivates hyc operon and Hyd-3 becomes major for H
2
produc-
tion at pH 7.5. This is in good conformity with our results with
high concentration of glucose (0.8%), when more formate
could be produced and thus activate hyc operon. As mentioned
above, Hyd-4 could be linked to the F
0
F
1
-ATPase supplying
reducing equivalents for energy transfer [19]. This means that
at limited conditions of glucose under fermentation Hyd-4
switches on involving in H
2
production. This might be argu-
mentative while proton motive force generated by wild type
cells has been determined to be higher than upon glycerol
fermentation at the same conditions where Hyd-2 was mainly
responsible for H
2
production [22]. Moreover, as it is known
Hyd-4 has more subunits than Hyd-2 and to drive Hyd-4
to produce H
2
more proton motive force generation is
required [22].
Besides, Skibinski et al. [23] did not detect any hyf depen-
dent H
2
production using hyaB hybC hycE triple mutant where
large subunits of Hyd-1, Hyd-2 and Hyd-3 were lacked. These
data have been obtained also in our study (data not shown)
while they exclude possible interaction between different Hyd
enzymes; each Hyd enzyme must work independently from
each other [23]. They do not consider that Hyd enzymes form
H
2
cycling and if this cycling is disturbed Hyd enzyme activity
might be changed. To confirm, it was shown also that the hycB
subunit of Hyd-3 is needed for H
2
production at pH 7.5 where
H
2
production was Hyd-4 dependent [9]. Moreover, compen-
satory uptake functions of Hyd-2 and Hyd-1 were shown by
Lukey et al. [24] and effects of hyb genes on hya expression
were established by the other authors [25].
Conclusions
It can be concluded that Hyd-4 is active mainly at low con-
centrations of glucose and at extreme pHs (pH 7.5 and pH 5.5).
Probably Hyd-4 switches on when there are glucose limited,
not reach conditions for cells. These results are of significance
for regulation of Hyd enzyme activity to enhance H
2
produc-
tion during fermentative conditions.
Acknowledgement
The authors thank Prof. Simon C. Andrews (The University of
Reading, Reading, UK) for supplying of mutants and valuable
advice. The study was supported by Research Grant of Min-
istry of Education and Science of Armenia to AT (#13F-002) and
Armenian National Science and Education Fund (USA) grant to
KT (#Biotech-3460).
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